Vol. 58, No. 2/2011 225–230 on-line at: www.actabp.pl Regular paper Cyclic enkephalin-deltorphin hybrids containing a carbonyl bridge: structure and opioid activity Małgorzata Ciszewska1, Katarzyna Ruszczyńska2, Marta Oleszczuk3, Nga N. Chung4, Ewa Witkowska1, Peter W. Schiller4, Jacek Wójcik2 and Jan Izdebski1* 1Peptide Laboratory, Department of Chemistry, University of Warsaw, Warszawa, Poland; 2Laboratory of Biological NMR, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warzsawa, Poland; 3Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada; 4Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, Quebec H2W IR7, Canada Six hybrid N-ureidoethylamides of octapeptides in which μ opiate receptor. However, its clinical utility is limited an N-terminal cyclic structure related to enkephalin was by the side effects, such as respiratory depression and elongated by a C-terminal fragment of deltorphin were development of tolerance and dependence. synthesized on MBHA resin. The synthetic procedure in- volved deprotection of Boc groups with HCl/dioxane and Enkephalins and other δ opiate receptor selective cleavage of the peptide resin with 45 % TFA in DCM. d- drugs share the morphin’s analgesic effect, but have re- Lys and d-Orn were incorporated in position 2, and Lys, duced negative properties: respiratory depression (Cheng Orn, Dab, or Dap in position 5. The side chains of the et al., 1993; Su et al., 1998) and reveal minimal potential dibasic amino function were protected with the Fmoc for the development of physical dependence (Cowan et group. This protection was removed by treatment with al., 1988). 55 % piperidine in DMF, and cyclization was achieved The development of new opioid peptides with in- by treatment with bis-(4-nitrophenyl)carbonate. Using creased selectivity for δ opioid receptor seems to be a various combinations of dibasic amino acids, peptides good avenue to obtaining drugs that may produce only containing a 17-, 18-, 19- or 20-membered ring struc- desired physiological responses. One of the methods ture were obtained. The peptides were tested in the that could allow reaching this goal is the design of con- guinea-pig ileum (GPI) and mouse vas deferens (MVD) formationally restricted peptides. The most promising assays. Diverse opioid activities were observed, depend- approach to obtaining a selective agonist is cyclization of ing on the size of the ring. Extension of the enkephalin a linear active peptide which can adopt a conformation sequence at the C-terminus by a deltorphin fragment re- able to interact preferentially with one receptor. This is sulted in a change of receptor selectivity in favor of the also expected to increase the enzymatic stability and ac- δ receptor. The conformational propensities of selected tivity of the resulting cyclic peptide. peptides were determined using the EDMC method in Peptides can be cyclized through formation of addi- conjunction with data derived from NMR experiments tional bonds, e.g., disulfide, lactone, lactame or forma- carried out in water. This approach allowed proper ex- tion of bridges between two amino-acid residues, e.g., a amination of the dynamical behavior of these small pep- carbonyl bridge or oligomethylene group (Davis, 2003; tides. The results were compared with those obtained Janecka & Kruszynski, 2005). In several cases incorpo- earlier with corresponding N-(ureidoethyl)pentapeptide ration of cyclized opioid peptides has been shown to amides. enhance selectivity for a distinct opioid receptor type. Further increase in selectivity of an active and selec- Keywords: cyclic opioid peptides, conformation, NMR, tive cyclic peptide is also possible as demonstrated by N-(ureidoethyl)amides, side-chain to side-chain cyclization; struc- replacement of cysteine by β,β-dimethylcysteine in an ture-activity relationship enkephalin analog cyclized by formation of a disulfide Received: 06 December, 2010, revised: 24 March, 2011; accepted: bridge (Akiyama et al., 1985). 08 May, 2011; available on-line: 17 May, 2011 Attempts to increase selectivity of a cyclic peptide by synthesis of chimeric peptides have also been reported: -Val-Gly-NH2, which is a C-terminal fragment of del- INTRODUCTION torphin address sequence (-Val-Val-Gly-NH2), was add- ed to the above-mentioned enkephalin analogs cyclized by formation of a disulfide bridge between two β,β- Endogenous opioid peptides, such as enkephalins, dimethylcysteine residues (Misicka et al., 1994). endorphins, deltorphins, dynorphins and endomorphins Previously we described the synthesis, biological activ- are involved in regulation of many biological processes, ity, and conformational analysis of several highly potent among them pain control. However, clinical utility as side-chain-to-side-chain cyclized analogs of enkephalin therapeutic analgesics is limited by their susceptibility to enzymatic degradation and several side effects. Biological * e-mail: [email protected] activities of opioid peptides are mediated through three Abbreviations: CLUST, a program for cluster analysis; EDMC, elec- major opioid receptor types (μ, δ and κ) and the distinct trostatically driven monte carlo; gHSQC, gradient heteronuclear structure of a peptide has an impact on the selectivity of single quantum coherence spectroscopy; MBHA, 4-methylbenzhy- receptor types and consequently on the activity profile. drylamine; MORASS, multiple overhauser relaxation analysis and simulation; ROESY, rotating frame overhauser enhancement spec- The analgesic effect of the most effective therapeutic troscopy; R.m.s.d., Root mean square deviation non-peptidic agent, morphine, is mediated through the 226 M. Ciszewska and others 2011 2 + + amides (H-Tyr-Gly-Gly-Phe-Met-NH2) in which Gly (M+Na ); 3: M calcld 993.4, obtained 1016.6 (M + Na ); and Met5 were replaced by dibasic amino acids and cyclic 4: M calcld. 951.3, obtained 974.5 (M+Na+); 5: M calcld. structure was obtained by incorporation of side-chain 965.5, obtained 988.6 (M+Na+); 6: M calcd. 993.4 ob- amino groups into urea residue (Pawlak et al., 2001; Filip tained 1016.4 (M+Na+). The structures of the peptides et al., 2005). Using the same synthetic procedure we ob- are presented in Fig. 1. tained analogs of an N-terminal segment of deltorphin (H-Tyr-Gly-Phe-Asp-) by introduction of dibasic amino Bioassays. The guinea-pig ileum (GPI) assay (μ re- acids in positions 2 and 4 (Filip et al., 2003). We also ceptor-representative) (Paton, 1957) and the mouse vas obtained N-(ureidoethyl)amides of enkephalin related to deferens (MVD) assay (δ receptor-representative) (Hend- the cyclic amides studied earlier, using a convenient syn- erson et al., 1972) were carried out as reported in de- thetic method developed in this laboratory (Ciszewska et tail elsewhere (Schiller et al., 1978; DiMaio & Schiller, al., 2009). 1980). A log dose-response curve was determined with [Leu5]-enkephalin as a standard for each ileum and vas Most peptides of those three series showed very high preparation and the IC50 values of the compounds being potency both in the guinea-pig ileum (GPI) and mouse tested were normalized according to a published proce- vas deferens (MVD) assays, which indicated a need to dure (Waterfield et al., 1979). The results are presented modify their structure to obtain more selective opiates. in Table 1. Addition of an address sequence of deltorphin (-Val-Val- NMR spectroscopy and theoretical analysis. The Gly-NH2) to the cyclic structure comprising positions methodology used for generation and selection of con- 1–4 resulted in greatly increased δ selectivity. Several formations was previously described for oxytocin and such peptides were a hundred times more active in the arginine-vasopressin (Liwo et al., 1996). It was applied by MVD assay than in the GPI assay (Zieleniak et al., 2008). us to one of the opioid peptides and described in de- In this work we obtained six hybrid peptides contain- tails in this journal (Sidor et al., 1999) and by others to ing a message sequence of enkephalin, restricted via a a number of compounds (Cohen et al., 2002; Masman urea bridge as described above, and a C-terminal address et al., 2006; 2008). Subsequently we used this method- of deltorphin. To facilitate the synthesis, the peptides ology with success in several studies of different opi- were obtained in the form of N-(ureidoethyl)amides. oid analogs (Pawlak et al., 2001; Filip et al., 2003; 2005; Ciszewska et al., 2009). In this paper NMR spectra of MATERIALS AND METHODS peptides 2, 3 and 4, which were sufficiently soluble in water, were recorded on a Varian INOVA 400 MHz and/or a Varian Unity PLUS 500 MHz spectrometer at Peptide synthesis. General procedure. The p-nitrophe- 25 °C in H2O/D2O (9 : 1, v/v). For 1D proton spectra noxycarbonyl derivative of Boc-diaminoethane was ob- 16k points were collected and a spectral width of 6 kHz tained as described earlier (Wiszniewska et al., 2005). was used. 2D experiments were measured collecting 2k This compound (0.401 g, 1.25 mmol) was coupled to points using 4.5 kHz spectral width in the proton direc- the MBHA resin (0.25 meq/g, 1 % crosslink, 100–200 tion and 256-point increments in the F1 direction. The mesh) in DMF at 60 ºC for 48 days. The Boc group same spectral width was applied in the F1 direction in was removed by treatment with 15 % HCl/dioxane, homo-nuclear 2D spectra, whereas a width of 25 kHz then Boc- amino acids were successively attached using and 2 kHz for carbon and for nitrogen, respectively, was N,N′-diisopropylcarbodiimide as a coupling reagent and used for hetero-nuclear correlations. A mixing time of 15 %HCl/dioxane for deprotection. The side chain ami- 0.08 s was used in TOCSY measurements and 0.25 s in no function was protected by Fmoc group. The Tyr side ROESY experiments. DSS was used as a reference exter- chain was not protected.